Multiplexed near infrared fluorescence lifetime imaging in turbid media

Abstract. Significance Fluorescence lifetime imaging (FLI) plays a pivotal role in enhancing our understanding of biological systems, providing a valuable tool for non-invasive exploration of biomolecular and cellular dynamics, both in vitro and in vivo. Its ability to selectively target and multiplex various entities, alongside heightened sensitivity and specificity, offers rapid and cost-effective insights. Aim Our aim is to investigate the multiplexing capabilities of near-infrared (NIR) FLI within a scattering medium that mimics biological tissues. We strive to develop a comprehensive understanding of FLI’s potential for multiplexing diverse targets within a complex, tissue-like environment. Approach We introduce an innovative Monte Carlo (MC) simulation approach that accurately describes the scattering behavior of fluorescent photons within turbid media. Applying phasor analyses, we enable the multiplexing of distinct targets within a single FLI image. Leveraging the state-of-the-art single-photon avalanche diode (SPAD) time-gated camera, SPAD512S, we conduct experimental wide-field FLI in the NIR regime. Results Our study demonstrates the successful multiplexing of dual targets within a single FLI image, reaching a depth of 1 cm within tissue-like phantoms. Through our novel MC simulation approach and phasor analyses, we showcase the effectiveness of our methodology in overcoming the challenges posed by scattering media. Conclusions This research underscores the potential of NIR FLI for multiplexing applications in complex biological environments. By combining advanced simulation techniques with cutting-edge experimental tools, we introduce significant results in the non-invasive exploration of biomolecular dynamics, to advance the field of FLI research.


Meital Harel, Uri Arbiv and Rinat Ankri
Ariel University, Faculty of Natural Science, Department of Physics, Ariel, Israel Content:     Figure S6 shows two representative lifetime results for the intensity pictures presented in Fig. 2.
These lifetime results are the foundation for the graph presented in Fig. 3f.We conducted simulations involving various optical properties, including a scattering coefficient that is an order of magnitude higher than the one presented in the paper (μs=503 cm-1 instead of μs=403 cm-1).The multiplexing results for the fluorescence lifetimes of the simulated samples at The two fluorophores presented the following optical properties: QY1 = 0.9%,  1 = 0.62 ns (yellow spot) and QY2 = 1.3%,  2 = 1 ns (magenta spot), with fixed separation distances, ∆ = 2 , as function of the tissues' thickness,  = 0: 1 are presented in Figure S6.(Results for z = 0.1,0.3cm are presented in Figure 4 in the paper).The ROI (white frame) for z = 0.5 -1 cm is 1  • 1  , whereas the ROI for z = 0 -0.1 cm is 3  • 3 .Phase FLT (nsec)

Figure S2 .Figure S4 .
Figure S2.The sliced tissue-like phantoms were used for the FLI experiments Figure S3.Average intensity <I> (measured in counts) for different depths z Figure S4.Mean lifetime values for different fluorophores corresponding to diverse depths Figure S5.intensity pictures of a single fluorophore located at a representative depth of z=0.3 cm Figure S6.Representative lifetime results for intensity pictures presented in Fig. 2.

Figure S7 .Figure S11 .Figure S12 .
Figure S7.Simulated fluorescence intensity and phasor analysis results, with higher scattering coefficient Figure S8.Simulated fluorescence intensities and lifetime histograms for two adjacent fluorophores Figures S9 and s10.Simulated fluorescence intensity and lifetime histogram with 1% cutoff for two adjacent fluorophores Figure S11.Extracted lifetimes versus depth: Figure S12.The decay profiles of ICG fluorescence emission vary across different phantom slices.

Figure S2 .Figure S3 .Figure S4 .
Figure S2.The sliced tissue-like phantoms were used for the FLI experiments.For each experiment, a slightly oversized slice of phantom was cut and placed on a phantom holder comprised of a glass coverslip bottom taped to the 3D printed spacer of appropriate thickness (0.1, 0.3, 0.5, 0.7 and 1 cm), then cut with a knife to achieve the desired thickness, and covered with another coverslip.
Figure S6.Representative lifetime results for intensity pictures presented in Fig. 2.These lifetime results are the foundation for the graph presented in Fig. 3f.

aFigure S7 .Figure S8 .Figure S9 .
Figure S7.Simulated fluorescence intensity and phasor analysis results, with higher scattering coefficient μs=503 cm-1, pertaining to two adjacent fluorophores: Two adjacent fluorophores (left spot -QY=0.9%,lifetime = 0.65 ns, right spot -QY=1.3%,lifetime = 1 ns) from a top-down view.The vertical separation between the centers of these fluorophores was ∆x = 2 cm, with an initial photons number a of ~7.6 • 10 7 .(a) The simulated intensity distribution, for a fluorophore's depth of 0.1 cm within the tissue.Dimensions are 2048*2048 pixels.The white square designates the ROI where phasor analyses were conducted (b) The histogram of lifetimes, for the large frame illustrated in Figure 1(a).(c) The histogram of lifetimes for the white frame of Figure 1(a), featuring a cutoff at 1%.

Figure S12 .
Figure S12.The decay profiles of ICG fluorescence emission vary across different phantom slices.

Figure S13 :
Figure S13: Extracting FLTs of IRDye800 imaged through intralipid tissue-like phantoms with thicknesses varying from 0 to 1cm.(a) The red circled dye is the IRDye800.Scale bar is 11mm.(b) Histograms for the phasor counts versus the lifetimes calculated from each phasor point.(d) The mean FLT for each phantom's thickness.(c) The mean FLT for each phantom's thickness.With the power of the microlensed SPAD512S, pixel-by-pixel background correction and phasor-based analyses, the FLT of the dye was extracted even behind a 1 cm phantom through the calculation of the mean of the phasor cloud.